Error prone DNA synthesis

Pannekoek H, van Meijer M, Schleef RR, Loskutoff DJ, Barbas CD, Functional display of human plasminogen-activator inhibitor I (PAI-1) on phages Novel perspectives for structure-function analysis by error-prone DNA synthesis, Gene, 128 135-140, 1993. 

Ponamarev, M. V., Longley, M. J., Nguyen, D., Kunkel, T. A., and Gopeland, W. C. (2002). Active site mutation in DNA polymerase gamma associated vdth progressive external ophthalmoplegia causes error-prone DNA synthesis. J. Biol Chem. 277, 15225-15228. 

Polr performs error-prone translesion synthesis opposite (+)- and (-)-trans-anti-BPDE-A -dG DNA adducts by predominantly inserting A opposite the lesion in vitro. This polymerase is more active in response to the former isomeric lesion. In yeast cells, Polr, Pol , and Revl are all required for G -> T transvertion mutations. The likely mechanism is A insertion opposite the lesion by Polr followed by extension synthesis by Pol . Revl probably plays a noncatalytic role in such a mutagenic bypass of the BPDE lesions. 

A, on the other hand, was not predicted to be genotoxic (i.e., a DNA-damaging agent), evidenced by its lack of potential to induce mutations in Salmonella, error-prone DNA repair, or unscheduled DNA synthesis in rat hepato-cytes (Table 19.5). Thus, even if the potential for murine carcinogenicity were accepted, in view of the fact that the vast majority of rec-... 

Li, Z., Zhang, H., McManus, T.P., McCormick, J.J., Lawrence, C.W., and Maher, V.M. (2002) hREV3 is essential for error-prone translesion synthesis past UV or benzojajpyrene diol epoxide-induced DNA lesions in human fibroblasts. Mutat. Res., 510, 71-80. 

The results of the Ames test for mutagenesis Indicate that many ruthenium compounds Introduce serious lesions into cellular genetic material so that an error-prone DNA repair mechanism is Induced. These results are similar to those obtained for clsplatln (M) and suggest that these complexes probably bind directly to nuclear DNA. In concert with this, many of the ruthenium complexes also Inhibit cellular DNA synthesis (H, ), another property also noted for the cls-platlnum drugs. Unfortunately, however, there Is no correlation between either of these studies and the antitumor activity of ruthenium compounds tested In animal systems. 

Use hot-start tubes and assemble the bottom and top part of the reaction for second-strand synthesis and amplification of the DNA template by error-prone PCR. Hot-start PCR is the PCR technique of assembling the reaction mixture at a temperature that is greater than the annealing temperature. This procedure increases precision, yield, and specificity. The pre-adhered wax bead assures synchronous reaction start-up and eliminates the need for using mineral oil. 

The Interaction of Replication Forks with DNA Damage Can Lead to Error-Prone Translesion DNA Synthesis... 

At a stalled bacterial replication fork, there are two avenues for repair. In the absence of a second strand, the information required for accurate repair must come from a separate, homologous chromosome. The repair system thus involves homologous genetic recombination. This re combinational DNA repair is considered in detail in Section 25.3. Under some conditions, a second repair pathway, error-prone translesion DNA synthesis (often abbreviated TLS), becomes available. When this pathway is active, DNA repair becomes significantly less accurate and a high mutation rate can result. In bacteria, error-prone translesion DNA synthesis is part of a cellular stress response to extensive DNA damage known, appropriately enough, as the SOS response. Some SOS proteins, such as the UvrA and UvrB proteins already described (Table 25-6), are normally... 

In bacteria, error-prone translesion DNA synthesis, involving TLS DNA polymerases, occurs in response to very heavy DNA damage. In eukaryotes, similar polymerases have specialized roles in DNA repair that minimize the introduction of mutations. 

DNA glycosylases 971 AP site 971 AP endonucleases 972 DNA photolyases 974 recombinational DNA repair 976 error-prone translesion DNA synthesis 976 SOS response 976 homologous genetic recombination 978... 

This test has several advantages in that primary liver cells have considerable activation capacity and the test measures an event at the DNA level. It does not, however, distinguish between error-free repair and error-prone repair, the latter being itself a mutagenic process. Thus it cannot distinguish between events that might lead to toxic sequelae and those that do not. A modification of this test measures in vivo unscheduled DNA synthesis. In this modification animals are first treated in vivo, and primary hepatocytes are then prepared and treated as already described. 

However, much data has been accumulated in recent years indicating that the replication machinery can elongate past cisplatin-DNA lesions in a mutagenic way [15], Intervention of specific DNA polymerases and protein-protein interactions between replicative enzymes and DNA damage-recognition proteins may lead to occasional translesion DNA synthesis. This translesion synthesis can occur in an error-prone fashion, leading to indue-... 

For a given lesion, error-free or error-prone synthesis by a bypass polymerase is often determined by in vitro translesion synthesis assays (Figure 22.21). The assay involves in vitro DNA synthesis by a purified bypass polymerase from an oligonucleotide template containing a site-specific lesion. A DNA primer labeled with 32P at its 5 end is annealed to the damaged template prior to assembling the assay reactions. Following the polymerase reaction, products are separated by electrophoresis... 

---